Vectors for genetic engineering of plants are highly desirable for the production of proteins, for endowing a host plant with a new trait, for suppressing a gene of the host plant, or for determining the function of a gene, notably a gene determined by genomics.
Vectors, notably viral vectors, for the genetic engineering of plants are already known. These must be capable of infection, amplification and movement (both cell-to-cell and long-distance) in a plant in addition to having at least one sequence for gene expression or suppression. Prior art vectors rely on subgenomic promoters as transcriptional elements. A subgenomic promoter has the effect that, in a transfected plant cell, transcription of a vector nucleic acid sequence starts in part at said subgenomic promoter to generate a shorter RNA so that translation of a gene downstream from said promoters by the plant translation machinery is enabled. Translation may then proceed cap-dependent. Such multiple transcriptions are kinetically disadvantageous because of waste of replicase capacity.
Such vectors have a number of further shortcomings. The introduction of a virus subgenomic promoter into a vector sequence makes said sequence longer and thus less efficient. Moreover, the presence of several identical or similar subgenomic promoters which are well adapted to transcription in the host gives rise to frequent recombination events and instability with loss of sequence portions. On the other hand, if significantly different subgenomic promoters are used, recombination may be suppressed but such promoters may be too different to be effectively recognized by the transcription system, which means loss of efficiency. Moreover, vectors are usually highly integrated entities with several interdependent functional elements or genes tightly packed into a sequence. This is the reason why the operability of a vector for certain heterologous genes or the like is somewhat idiosyncratic and frequently gives unpredictable results, notably in terms of infectivity and expression. Further, the available sequence space for promoters is usually constrained if sequence overlaps with upstream genes are present.
Therefore, it is an object of this invention to provide a novel vector for plant genetic engineering which is capable of efficient and stable operation in a host plant. It is a further object to provide a vector which is capable of high-level expression of a gene in a plant.
It has been surprisingly found that these objects can be achieved with a vector capable of amplification and expression of a gene in a plant comprising a nucleic acid having a sequence for at least one non-viral gene to be expressed and having or coding for at least one IRES element necessary for translation of a gene downstream thereof.
It has been previously suggested (WO 98/54342) to use a plant IRES element in a recombinant DNA molecule that has merely the function of gene expression (after integration into the host genome). However, the expression level is low. The exact reasons for this low expression level are not known. In any event, expression is limited to the very plant cells transformed, thus the overall efficiency in whole plants is extremely low.
It has been surprisingly found that it is possible to construct a plant vector which, when introduced into a plant cell, has not only the capability of gene expression but which has several additional functions which are all required for amplification and spreading throughout the plant so that the overall efficiency is extremely high. These functions comprise infection, amplification, cell-to-cell movement and long-distance movement. It is surprising that the required high degree of integration of functional and structural elements on a vector does not impair gene expression from said vector.
The IRES element of said vector can be located upstream of said non-viral gene to be expressed for directly supporting its translation. Alternatively, said IRES element may indirectly support the translation of said gene to be expressed by directly supporting the translation of another gene essential for a function of said vector selected from the group of infection, amplification, virus assembly, ability to suppress the silencing of viral infection development in plant cells, ability to redirect the metabolism in plant cells, and cell-to-cell or long-distance movement of said vector and cell-to-cell or long-distance movement of said vector.
Further said vector may comprise at least a portion of a sequence of the host plant genome in an anti-sense orientation for suppressing a gene of the host plant.
It is a further object to provide a vector which is capable of the effective suppression of a gene in a plant. This object has been achieved by a vector capable of amplification in a plant comprising a nucleic acid having or coding for at least one IRES element necessary for translation of a gene required for amplification of said vector and located downstream of said IRES element, said vector further comprising at least a portion of a sequence of the host plant genome in an anti-sense orientation for suppressing a gene of the host plant.
Further preferred embodiments are defined in the subclaims.
Here, the first plant expression and amplification vectors based on plant active translational (IRES) elements are described. Existing IRES elements isolated from animal viruses do not support translation in plant cells. Therefore, knowledge accumulated in animal expression systems is not applicable to plants. Animal IRES elements have never been tested for other functional properties, such as residual promoter activity, so this invention discloses the first bona fide cases of gene expression in plants relying exclusively on translation rather than on transcription with a subgenomic promoter necessary for expression of a gene downstream thereof.
The vectors of this invention allows preferably for regulation and preferential expression of a gene of interest in a plant by suppressing cap-dependent translation. In another preferred embodiment, very short homologous or artificial IRES elements are used, thus adding to the stability of the resulting vectors.
A preferred advantage of this strategy is that IRES sequences can be inserted upstream or downstream of viral gene(s) (e.g. the coat protein gene of tobacco mosaic virus such that translation of downstream foreign gene(s) or the viral gene(s), respectively, may occur via cap-independent internal ribosome entry pathway. Thus, said cap-independent translation of foreign gene(s) will occur from bicistronic or/and polycistronic RNAs.